Process for the isolation and purification of a glycoprotein avidin

ABSTRACT

The present invention provides a process for the preparation of Avidin having a high purity of 98% from egg white, the said process comprising lyophilizing the homogenized egg white with buffer followed by equilibrating the lyophilized material with cation exchanger for an hour at a temperature range of 20° to 30° C.; filtering the mixture to obtain a filtrate and matrix residue; washing the matrix twice with buffer; separating the washings to obtain washed matrix; adding 0.01 M dye in buffer to the washed matrix; equilibrated for an hour at an ambient temperature; separating the supernatant; adding acetic acid to supernatant and dialysing the solution till it is decolorised to obtain pure Avidin.

TECHNICAL FIELD

[0001] The present invention provides an economical and an easily operable process for the preparation of “Avidin” a glycoprotein from egg white using cation exchanger and HABA (Hydroxyazobenzene-2′-carboxylic acid) as a selective eluant.

BACKGROUND ART

[0002] Avidin is a glycoprotein, which occurs in egg white and is well known because of its property of binding to biotin, with an association constant of 10¹⁵ M⁻¹, the highest known for a bioaffinity pair.

[0003] Eatkin et al (1940) and Pennington et al. (1942) reports presence of Avidin and isolation in crude form. Conrat et al. (1952) reports very tedious and multi-step process for the isolation of Avidin by involving salt precipitation and adsorption on Bentonite. The above references are pertaining to early days when Avidin was not even characterized at the molecular level.

[0004] N. M. Green (1965) screened several anionic dyes for studying binding to Avidin and examined spectroscopically. The most striking results were obtained by using 4′-hydroxy azobenzene-2-carboxylic acid (HABA). Green et al. (1970) describes a multi-step process for the isolation of Avidin involving considerable amount of time. Bayer and Wilchek, (1974) used affinity chromatography on biocytin (ε-N-biotinyl-L-lysine)-linked matrices for the purification of Avidin. Since the association constant with biocytin is very high,

[0005] 6-M guanidine hydrochloride of pH 1.5 was used for elution. This resulted in poor and irreproducible results. Wilchek and Bayer, (1988) have described Avidin in enzyme-linked immunosorbent assay (ELISA), immunochemical staining, electron microscopy and affinity chromatography.

[0006] U.S. Pat. No. 4,966,851 describes the isolation of Avidin using weakly acidic ion-exchanger resin from egg-white by employing stepwise elution with buffer to obtain Avidin having a very low purity of about 12%. Thus, the process leads to non-specific recovery of target protein.

[0007] All the prior art processes described earlier involve either multi-step isolation or poor purity of Avidin. The present invention describes a quick, economical, efficient and a single step process for the isolation of Avidin from egg white. Also, the present process has a distinct feature of HABA being used as an eluant to carry out desorption of Avidin from the solid matrix cation exchanger used in the isolation process in comparison to the prior art processes wherein HABA has been always covalently bonded to the solid matrix and the covalently bonded conjugate is in turn used in the process for the isolation of Avidin.

OBJECTS OF THE INVENTION

[0008] The main object of the invention is to provide a process for the isolation and purification of Avidin from egg white in a single step.

[0009] Another object of the invention is to develop economical and easily operable process for the isolation and purification of Avidin.

[0010] Still another object of the invention provides a process of obtaining high purity Avidin from egg white.

[0011] Still another object of the invention is to provide a method wherein cation exchanger used can be recycled several times in the process.

[0012] Still another object of the invention is to provide a process wherein covalent binding of HABA is not encountered with the cation exchanger used.

[0013] In yet another objective of the invention is to develop commercially feasible process for the isolation of Avidin from egg white.

[0014] One more objective of the invention is to develop a process wherein some of the raw materials used are recycled.

SUMMARY OF THE INVENTION

[0015] Accordingly, the present invention provides an economical and easily operable process for the preparation of Avidin having high purity from egg white.

DETAILED DESCRIPTION OF THE INVENTION

[0016] In accordance to the objectives of the invention, the present invention provide a process for the preparation of Avidin having a high purity of 98% from egg white, the said process comprising steps of:

[0017] a) homogenizing egg white with buffer,

[0018] b) lyophilizing homogenized solution of step (a) to obtain a solid material,

[0019] c) dissolving solid egg white in buffer,

[0020] d) adding solution of step (c) to equilibrated cation exchanger incubation for an hour at temperature range of 20° to 30° C.,

[0021] e) filtering the mixture of step (d) to obtain a filtrate and matrix residue,

[0022] f) washing matrix residue of step (e) twice with buffer, removing the washing to obtain washed matrix,

[0023] g) adding 0.01 M dye in buffer to the washed matrix of step (f) and equilibrated for an hour in orbital shaker at an ambient temperature;

[0024] h) removing the supernatant of step (g),

[0025] i) adding acetic acid to supernatant of step (h), so that final concentration of acetic acid is 0.2 N in the solution,

[0026] j) dialysing the solution of step (i) till it is decolorized to obtain pure Avidin

[0027] In an embodiment, Avidin having a purity of 98% was obtained.

[0028] In another embodiment, the Avidin is estimated by conventional calorimetric dye-binding assay.

[0029] In still another embodiment, the buffer used is selected from acetate buffer or citrate buffer.

[0030] In still another embodiment, the concentration of the buffer used is in the range of 0.05 M to 0.15 M and preferably 0.1 M.

[0031] Yet another embodiment, the acetate buffer used has a pH in the range of 5.0 to 6.0 and preferably 5.5.

[0032] In another embodiment, the citrate buffer used has a pH in the range of 5.0 to 6.0 and preferably 5.5.

[0033] In still yet another embodiment, the dye used is selected from HABA, 4-hydroxy azobenzene-4-carboxylic acid or 4-dimethylaminobenzene-2-carboxylic acid.

[0034] In still another embodiment, the solid matrix used for purification is cationic exchanger. In still another embodiment, the cation exchanger used is selected from a group consisting of Streamline SP (Amersham Pharmacia, Sweden), CM cellulose (Whatmann, UK) or CM-Sephadex (Pharmacia, Sweden), SP Sephadex, Seralite SRC1120, Seralite WRC-50 and preferably Streamline SP (Amersham Pharmacia, Sweden). The invention is further illustrated by following examples, which should not be construed to limit the scope of this invention.

EXAMPLE 1

[0035] Assay of Avidin and Protein

[0036] The amount of Avidin in egg white and various fractions is estimated by the colorimetric dye-binding assay (Green, 1970b). The assay is based on the displacement of HABA from the dye-Avidin complex on the addition of biotin and the consequent decrease in absorption of the test solution at 500 nm. The concentration of Avidin (in mg ml⁻¹) was calculated as 0.49×ΔA₅₀₀.

[0037] The amount of protein is measured by the standard dye-binding assay, using bovine serum albumin as the standard protein.

EXAMPLE 2

[0038] Isolation of Avidin from Egg White

[0039] The whites of four eggs were separated and homogenized. The homogenized solution was lyophilized to obtain about eight grams of solid material. This was dissolved in 25 ml of 0.1 M acetate buffer, pH 5.5 and used as the crude source of avidin.

EXAMPLE 3

[0040] Purification of Avidin on a Cation Exchanger in the Batch Mode

[0041] Streamline SP (5 ml) was equilibrated with 50 ml 0.1 M acetate buffer, pH 5.5. After, equilibration, the excess buffer is drained completely and the dissolved egg white (25 ml) is added to the cation exchanger and allowed to equilibrate for one hour at 25° C. The unbound egg white solution is removed and the matrix is washed twice with 50 ml of 0.1 M acetate buffer, pH 5.5. The supernatant and the washings were measured for avidin activity and amount of protein. The excess buffer was then removed and 20 ml of 0.01 M HABA solution (in 0.09 M acetate buffer, pH 5.5) is added to the avidin-bound matrix and the solution allowed to equilibrate for one hour at 25° C. (100 rpm, on an orbital shaker). After one hour, the supernatant is collected. Acetic acid (830 μl of 5 N stock solution) is added to dissociate the complex of HABA and avidin. The solution is dialyzed extensively against the assay buffer till the solution becomes colorless. This solution is then assayed for avidin and amount of protein.

MAIN ADVANTAGES OF THE INVENTION

[0042] 1) the process is economical and easy to operate for isolating bulk quantity of avidin.

[0043] 2) the process provides high purity of avidin.

[0044] 3) provides a process for selective elution of avidin with HABA 

1) a process for the isolation and purification of avidin from egg white, the said process comprising steps of: a) homogenizing egg white with buffer, b) lyophilizing homogenized solution of step (a) to obtain a solid material, c) dissolving solid egg white in buffer, d) adding solution of step (c) to equilibrated cation exchanger and incubating for an hour at a temperature range of 20° to 30° C., e) filtering the mixture of step (d) to obtain a filtrate and matrix residue, f) washing matrix residue of step (e) twice with buffer, removing the washing to obtain washed matrix, g) adding 0.01 M dye in buffer to the washed matrix of step (f) and equilibrated for an hour in orbital shaker at an ambient temperature; h) removing the supernatant of step (g), i) adding acetic acid to supernatant of step (h), so that final concentration of acetic acid is 0.2 N in the solution, and j) dialysing the solution of step (i) till it is decolorised to obtain pure Avidin. 2) A process as claimed in claim 1, wherein in steps (a), (c), (f) and (g) the buffer used is selected from group consisting of acetate buffer or citrate buffer. 3) A process of claim 2, wherein in the acetate buffer used is prepared by mixing a solution of sodium acetate and acetic acid. 4) A process of claim 2, wherein citrate buffer used is prepared by mixing a solution of sodium citrate and citric acid. 5) A process of claim 3, wherein the acetate buffer used has a pH in the range of 5.0 to 6.0 and preferably 5.5. 6) A process of claim 4, wherein the citrate buffer used has a pH in the range of 5.0 to 6.0 and preferably 5.5. 7) A process as claimed in any of the preceding claims, wherein the concentration of the buffer used is in the range of 0.05 M to 0.15 M and preferably 0.10 M. 8) A process as claimed in claim 1, wherein in step (d) the cation exchanger used is selected from a group consisting of Streamline SP (Amersham Pharmacia, Sweden), CM cellulose (Whatmann, UK) or CM-Sephadex (Pharmacia, Sweden), SP Sephadex, Seralite SRC-1120, Seralite WRC-50 and preferably Streamline SP (Amersham Pharmacia, Sweden). 9) A process as claimed in claim 1, wherein in step (g) the ionic strength of the equilibration buffer and a solution of HABA in equilibration buffer used are preferably the same. 10) A process as claimed in claim 1, wherein in step (g) the dye used is an azo dye. 11) A process of claim 10, wherein the azo dye used is selected from a group consisting of HABA, 4′-hydroxyazobenzene-4-carboxylic acid or 4′-dimethylaminobenzene-2-carboxlic acid and preferably HABA. 12) A process as claimed in claim 1, wherein in step (j) the dialysis is performed at a temperature range of 0° C. to 5° C. and preferably 4° C. 13) A process as claimed in claim 1, wherein in step (j) the Avidin obtained has a purity of 98%. 14) A process as claimed in claim 1, wherein most of the steps are performed at an ambient temperature. 